尿液嗜中性白血球明膠酶相關運載蛋白之分泌與轉譯後修飾對體染色體顯性多囊腎病細胞與囊泡之影響

Abstract

多囊性腎臟疾病（Polycystic kidney disease, PKD）是最常見的腎臟染色體遺傳疾病，主要可分為體染色體顯性多囊性腎臟病（ADPKD）與體染色體隱性多囊性腎臟病（ARPKD）兩型。ADPKD主要是PKD1基因與PKD2基因，少數由GANAB基因突變所引發，促使腎小管上皮細胞異常增生。PC1和PC2形成複合體調節Ca2+離子通道，改變細胞內Ca2+離子濃度，進而影響下游訊息傳導途徑。此疾病之病徵為腎臟有充滿液體的囊泡（cyst）堆積，隨著疾病進程增加，囊泡體積的擴大會壓迫腎臟周圍組織，最終會導致腎功能的衰竭。目前，美國食品藥物管理屬（U.S. Food and Drug Administration, FDA）唯一核准ADPKD用藥Tolvaptan，做為vasopressin V2 receptor的拮抗劑來抑制囊泡生長，然而藥物伴隨肝毒性之風險以及昂貴的金額支出。實驗室先前研究發現，在ADPKD（Pkd1L3/L3）模式小鼠試驗（in vivo）、細胞試驗（in vitro）上透過基因技術使ADPKD鼠在腎臟中大量表現或細胞內大量產生嗜中性白血球明膠酶相關運載蛋白（NGAL），皆可減緩囊泡增大速度。NGAL是一個22 kDa的分泌型蛋白，在臨床上為腎損傷之指標。本研究結果顯示，過度表達分泌型和非分泌型mNgal單株細胞均可減緩囊泡增大。然而，目前還沒有足夠的證據表明其對ADPKD的作用機制。我們的研究也另外揭示了ADPKD細胞中，過度表現分泌型Ngal可透過促進細胞凋亡與上調Rb信息傳導途徑抑制細胞增生。此外，ADPKD細胞在添加帶有轉譯後修飾之倉鼠卵巢細胞 (Chinese Hamster Ovary, CHO）所生產的mNgal蛋白以及含有mNgal的條件培養基後，對囊泡生長具有緩和效果。希望這些成果未來可以為ADPKD提供新的治療策略。Polycystic kidney disease (PKD) is the most common chromosomal genetic disease of the kidney, which can be mainly classified into autosomal dominant polycystic kidney disease (ADPKD) and recessive polycystic kidney disease (ARPKD). ADPKD is mainly caused by the mutation of PKD1 (85 %) and PKD2 (15 %) genes, and a few are caused by GANAB genes, which causes the renal tubular epithelial abnormal proliferation. PKD1 and PKD2 encode polycystin-1 (PC1) and polycystin-2 (PC2) proteins, respectively. PC1 and PC2 form a complex to regulate Ca2+ ion channels and affect many downstream Ca2+ related signaling pathways. The disease is characterized by the accumulation of fluid-filled cysts on the kidneys. As the disease progresses, the enlargement of the cysts can compress tissues around the kidney and eventually lead to kidney failure. Currently, the only drug that U.S. Food and Drug Administration (FDA) approved for ADPKD, Tolvaptan, blockage of vasopressin V2 receptor to inhibit the growth of cysts. However, the drug is associated with high liver toxicity and high cost. Neutrophil gelatinase-associated protein (NGAL) is a 22kDa secreted protein that is clinically used as an acute and chronic kidney injury marker. Our previous studies showed that overexpressed Ngal on ADPKD (Pkd1L3/L3) model mouse (in vivo) and cell line (in vitro) could inhibit cell proliferation and alleviated cysts progression. Our results showed that, overexpressed both secreted and non-secreted mNgal single clonal cells can decrease cyst progression. However, there is not enough evidence to reveal the mechanism of the Ngal for the treatment of ADPKD disease yet. Our study also uncovers that overexpression of secreted Ngal can inhibit cell proliferation by promoting apoptosis and upregulating the Rb signaling pathway in ADPKD cells. Furthermore, both addition of mNgal protein produced from mammalian CHO cell and conditional medium (CM) which containing mNgal with post-translational modification (PTM) reduced the cyst growth in the ADPKD cell model. We hope these results might provide a therapeutic strategy for ADPKD in the future.